The goal of this work is to create a system that will produce site-specific integration of gene therapy vectors into the chromosomes. This feature is currently lacking in all cases. The innovative system developed here will provide long-term expression of introduced genes, for the lifetime of the target cells. This outcome is desirable in most gene therapy strategies. In order to achieve efficient and site-specific integration, Dr. Calos will use prokaryotic recombinase enzymes that work in mammalian cells with no added co-factors. These enzymes have long recognition sites that are not expected to occur in mammalian genomes. However, the principal investigator has shown that enzymes also recognize a small number of native sequences, called pseudo sites, with homology to the native recognition site. These pseudo sites can be used for site-specific integration into mammalian chromosomes. The principal investigator will locate such sites for three recombinase enzymes, using integration efficiency and good gene expression in a chromosomal context. Protein engineering will be carried out on the recombinases to improve efficiency and alter DNA recognition specificity. Gene therapy experiments will be initiated with the best enzyme/pseudo site candidates by using introduction of plasmid DNA. The novel site-specific integration strategy developed here can be used to add value to most current gene therapy vectors.